PROJECT SUMMARY/ABSTRACT The increased accessibility of high-throughput screening methods and instruments has revolutionized how the scientific community searches for small molecules that modulate protein function and cellular pathways. The primary goal of mining large collections of small-molecule libraries against in vitro and cellular assays is to identify compounds for the development of chemical tools to manipulate biological systems with spatial, temporal, and concentration-dependent control, as well as provide critical information on drug development. Unfortunately, most initial screening hits require extensive optimization to yield molecules with the necessary potency and selectivity for biological applications. Lead optimization is a significant hurdle in the drug and probe development process due to the costly amounts of consumables, labor expenses and time, and reliance on the intuition and synthetic skills of medicinal chemists. Critically, a majority of academic labs do not have access to medicinal chemistry and often use low-quality chemical probes that may provide misleading results. The design of a universal and cost-effective high-throughput medicinal chemistry platform is therefore a significant priority. My application is focused on the development, validation, and use of a practical, comprehensive platform to synthesize lead compound analog library and test biological activity in an high-throughput manner. I have recently repurposed sulfur(VI) ?uoride exchange (SuFEx) reactions between iminosulfur oxydi?uoride (?isodifluor?)-containing molecules and amines that yield >80% of the desired product in DMSO and PBS. My exciting initial studies show that I can synthesize a focused libraries of lead compound analogs on picomole scale, directly assess the products with in vitro assays, and develop drug-like ligands with improved biological functions. Here, my goals are to: 1) develop, streamline and validate the platform (SA1); 2) rigorously assess the comprehensiveness of my platform against a panel of protein targets with diverse in vitro and cell-based assays (SA2); and 3) apply the platform to functional fragment molecules to identify protein targets with novel biological functions. The successful completion of this highly collaborative study among the Wolan, Sharpless, Cravatt, and Calibr will provide a robust medicinal chemistry platform for the research community. Chemical probes developed by the platform will ultimately improve our molecular-level understanding of fundamental biological processes and discovering approaches to their control.